The invention concerns a method for replacement of a homologous gene segment from mammals in the cell line of nonhuman mammals by homologous recombination. The invention also concerns a method for creation of a transgenic nonhuman mammal, as well as its use for expression of gene products and for testing of drugs and therapeutic models. In addition, a recombination vehicle for homologous recombination, a stably transfected cell clone and a transgenic, nonhuman mammal are disclosed.
The field of this invention is the production of humanized antibodies in a transgenic host.
Monoclonal antibodies find application in both diagnosis and treatment. Because of their capacity to bind to a specific epitope, they can be used to identify molecules carrying that epitope or may be aimed, by themselves or in conjunction with another moiety, to a specific place for diagnosis or therapy. Humanized antibodies posess significant advantages over rodent antibodies, however they have been difficult to produce in large quantities.
Various technologies have been developed to overcome problems related to the production of human monoclonal antibodies, one strategy is the generation of chimeric antibodies in which the rodent constant (C) regions of both heavy (H) and light (L) chains, with or without the framework of the variable region, are replaced by the equivalent domains or sequences of human immunoglobulin. Another strategy attempts to mimic the immune response in vitro, through bacteriophage expression of human variable region genes isolated from human B cell populations, followed by selection for rare, high affinity antibodies through antigen binding. A major drawback to these and similar approaches is the cumbersome work required to generate each specific mAb of appropriate biological function.
An ideal solution to these problems would be the generation of a mouse strain synthesizing human antibodies instead of mouse antibodies. This has been approached by introducing a mini-locus containing a few human V and C region gene segments in germline configuration into the mouse genome as a transgene. In such strains, antibodies carrying human H and L chains were indeed produced, but the levels of production were low and the repertoire of human V regions was severely limited. Thus, while the approach appears promising in principle, it is not yet at the stage to stand its final test. There is, therefore, substantial interest in finding alternative routes to the production of allogeneic antibodies for humans.
Relevant Literature
Homologous recombination between the DNA sequences present in a chromosome and new, added, cloned DNA sequences (hereafter referred to as gene targeting) permits insertion of a cloned gene into the genome of a living cell. Animals that are homozygous for the desired mutation can be obtained with this method using embryonal germ cells via chimeras (M. R. Capecchi, Science, 244, 1288 (1989)). The use of gene targeting to deactivate a gene (gene disruption) and for gene correction, i.e., incorporation of a gene segment previously not present, is described in R. D. Camerini-Otero, R. Kucherlapati, The New Biologist, 2(4), 334-341 (1990).
The insertion methods described in WO 90/11354 and WO 91/19796 in which a desired gene segment is introduced into the genome of a cell by homologous recombination are also included among the gene correction methods. In the latter method the still functional endogenous gene is removed in a second step and an endogenous gene segment thus replaced with a homologous gene segment. Moreover, WO 91/19796 discloses a virtually one-stage method, i.e., co-transfection, in which the resistance marker is not situated in the gene segment being introduced, but is introduced separately. In the examples cited in this document, however, only slightly varied homologous gene segments (maximum 2xc3x972 base variations) are introduced, raising the question as to the extent to which a selectable recombination event can still be established during a reduction in homology (given the limited recombination frequency of the two-stage method). Nor is it demonstrated in this document whether the executed mutation of the embryonal parent cells is transferred to the cell line. Moreover, K. Rajewsky (Science, 256, 483 (1992)) suggests the use of homologous recombination for gene substitution in order, for example, to identify the function of a newly discovered gene.
In this sense, the task of the present invention was to make available a direct successful method in one step for production of genetically engineered nonhuman mammals that contain homologous gene segments from other mammals via homologous recombination.
Homologous recombination between the DNA sequences present in a chromosome and exogenous DNA sequences permits insertion of a cloned gene into the genome of a living cell. The generation of transgenic animals using this methodology is described in Thomas and Capecchi (1987) Cell 51:503-512; Capecchi (1989) Science 244:1288 and Koller and Smithies (1989) P.N.A.S. 86:8932-8935. The use of gene targeting for gene correction is described in Camerini-Otero and Kucherlapati (1990) The New Biologist 2:334-341. Targeted deletion of gene segments using the bacteriophage-derived Cre-loxP recombination system is described in Sauer and Henderson (1988) P.N.A.S. 85:5166-5170 and Orban et al. (1992) P.N.A.S. 89:6861-6865. K. Rajewsky (1992) Science 256:483 suggests the use of homologous recombination for gene substitution in order to identify the function of a newly discovered gene. Yung et al. (1993) Science 259:984-987 describe the generation of transgenic animals using the Flp/frt recombinase system.
WO 90/11354 and WO 91/19796 further describe methods of homologous recombination. In the former, a functional endogenous gene is removed in a second step after homologous recombination, thereby replacing an endogenous gene with a homologous gene segment. WO 91/19796 discloses a method in which the resistance marker is not situated in the gene segment being introduced, but is introduced separately.
The genes encoding human and mouse immunoglobulins have been extensively characterized. Berman et al. (1988) EMBO J. 7:727-738 describe the human Ig VH locus. Sakano et al. (1981) Nature 290:562-565 describe a diversity segment of the immunoglobulin heavy chain genes. Blankenstein and Kruwinkel (1987) Eur. J. Immunol. 17:1351-1357 describe the mouse variable heavy chain region.
The generation of transgenic mice bearing human immunoglobulin genes is described in International Application WO 90/10077 and WO 90/04036. WO 90/04036 describes a transgenic mouse with an integrated human immunoglobulin xe2x80x9cminixe2x80x9d locus. WO 90/10077 describes a vector containing the immunoglobulin dominant control region for use in generating transgenic animals.
Animals, DNA compositions and methods are provided for the efficient production of high affinity humanized antibodies. Transgenic animals are produced through targeted gene replacement. The native immunoglobulin constant region is replaced with the corresponding human gene segment. Of particular interest is the use of non-mammalian recombinase systems in embryonic stem (ES) cells, which allows for a convenient replacement process. Humanized antibodies are made at a high level and efficiency. In a preferred embodiment, transgenic animals are obtained that undergo antibody affinity maturation and a class switch from the native immunoglobulin to the humanized form. A method was found by the applicant that permits targeted replacement of individual gene segments in the cell line of a mammal in one step with gene segments of other species.
The present invention thus concerns a method for replacement of a gene or gene segment in the cell line of a nonhuman mammal with a homologous gene or a homologous gene segment of another mammal, in which (i) an embryonal parent cell line is transfected with a selectably marked recombination vehicle; (ii) stably transfected cell clones are selected for the presence of the marker gene; (iii) they are subjected to targeted selection by PCR and/or Southern Blot; (iv) these are injected into the blastocysts of the nonhuman mammal; (v) the blastocysts are transferred to surrogate mothers, characterized by the fact that the endogenous gene or the endogenous gene segment is functionally replaced in one step by the homologous gene or the homologous gene segment in the recombination event by means of the selectably marked recombination vehicle.
It is preferred according to the present invention that the introduced gene or the introduced gene segment originate from humans and that the nonhuman mammal be a rodent, especially a mouse.
Genes or gene segments that code for proteins involved in the immune system, the nervous system, especially signal-mediating and adhesion molecules, virus receptors, the blood-forming system and support tissue, especially muscles, tendons and bones may also be replaced according to the subject methods. Those genes and gene segments that code for protein of the immune system are particularly preferred, especially antibody genes, T-cell receptor genes, cytokines, cytokine receptor genes, MHC genes, adhesion molecule genes and genes of signal-mediating molecules.
An antibody gene segment of the mouse is replaced by an antibody gene segment of man in a special variant of the present invention.
The selectably marked recombination vehicle according to the invention is a replacement vector and carries the gene or gene segment to be introduced, sequences that are homologous to the sequences that flank the endogenous DNA segment to be replaced and a marker gene, especially neomycin or hygromycin, neomycin being preferred. Moreover, the recombination vehicle can also contain viral recognition sequences (for example SV40), additional sequences to amplify gene expression, target sequences for pro- and eukaryotic recombination systems. The latter sequences, especially the Cre recognition sequence LoxP or the flip recognition sequence-Frt, can be used for targeted removal of marker genes, as well as any still remaining non-functional target gene segments. In this fashion it is possible to replace in the first step the endogenous gene segment with a homologous gene segment of another mammal and to remove the remaining residues in a second step by means of a selectively functioning recombinase (H. Gu et al., Cell, 73, 1155-1164 (1993)). The remaining residue is selectively removed in this method and only the desired recombination can occur, in contrast to the xe2x80x9chit and runxe2x80x9d method described in WO 91/19796.
The selectably marked recombination vehicle in a preferred variant of the present invention is the plasmid pTZ2-CkN (PAxe2x88x92)(DSM 7211).
Another object of the present invention is a recombination vehicle for homologous recombination that contains the gene to be replaced or the gene segment to be replaced and a selectable marker gene. The recombination vehicle can also contain the recognition, amplification and/or target sequences already mentioned.
Another object of the present invention is the stably transfected cell clone produced by the method according to the invention, as well as a method for creation of a transfected, nonhuman mammal. According to the latter method the stably transfected cell clones according to the invention are injected into mouse blastocysts, these blastocysts are transferred to the surrogate mother, the born chimeral animals are mated and their offspring selected for the presence of the mutation.
Transgenic nonhuman mammals that can be obtained in this fashion are also an object of the present invention.
Another object of the present invention is the use of the selectively mutated, transgenic nonhuman mammal for expression of gene products of another mammal instead of the product coded by the original gene segment and for testing of drugs and therapeutic models. The use of these gene products to produce humanized monoclonal antibodies and for virus production is particularly preferred in the sense of the present invention.
The method according to the invention permits replacement of genes or gene segments in the cell line of nonhuman mammals with a homologous gene or a homologous gene segment of another mammal in one step. Animals that are homozygous for the desired mutation are obtained via chimeras and can be used for expression of gene products of another animal instead of the endogenous gene or for testing of drugs and therapeutic models.